The overall goals of this proposal are to understand the three dimensional structures of the amyloid Abeta peptide as soluble low molecular weight species, oligomeric intermediates and when it is polymerized into amyloid fibrils. The goal is also to understand the dynamics of Abeta assembly into amyloid fibrils and the mechanisms of action of inhibitors of amyloid assembly. Previous work on the effects of mutations associated with familial forms of AD on the production of Abeta implicates amyloid as a causative agent in the etiology of AD. Results from studies on Abeta toxicity in cell culture systems suggest that the structure and assembly state of Abeta also plays a critical role in the biological activity of Abeta. The first specific aim is to determine the three dimensional structures of Abeta in solution and when it is assembled into amyloid fibrils. Distances between different points along the Abeta sequence will be measured in the soluble, oligomeric and fibrillar state by site-directed spin labeling and electron paramagnetic resonance (SDSL/EPR) spectroscopy and solid state nuclear magnetic resonance SS-(NMR). The conformation and location of solvent exposed amino acid residues and positions residing in polar and non-polar environments will be probed by SDSL/EPR. This information will be used to construct three-dimensional models of Abeta. We will examine the structural basis for potential alternative structures in "nonfibrillar" insoluble Abeta that are believed to be less pathologically significant than fibrillar Abeta. In the second specific aim, we will characterize soluble oligomeric aggregates that may represent key nucleation centers or intermediates in the fibril, assembly reaction and determine which steps are rate limiting. These results will also establish the stabilities of the intermediates and identify rate-limiting steps in the assembly, process. The issue of whether fibril assembly is bi-directional will provide an independent means of evaluating models of fibril structure. In the third aim, we will examine the mechanism of action of a number of different inhibitors of amyloid aggregation. We propose to characterize how these molecules interact with Abeta and determine which step(s) of assembly these molecules inhibit. This information may ultimately be useful in conceptualizing strategies and therapeutic agents to interfere with amyloid assembly and deposition in vivo and in understanding the relationship between Abeta structure and cytotoxic activity.